Pharmaceutical combinations comprising corticoids and immunosuppressants for treating corticoid- and/or calcineurin inhibitors-resistant diseases

ABSTRACT

The use of a combination of a corticoid and a compound of formula I for the preparation of a medicament for the treatment of a corticoid-resistant disease and/or a calcineurin inhibitor-resistant disease, e.g. where T cells are involved in the pathophysiology.

The present invention relates to new uses of pharmaceuticalcombinations, e.g. combinations of pharmaceutically active compounds.

Substances that inhibit T-cell proliferation, i.e. proliferation of Tlymphocytes, have been used therapeutically as immunosuppressive and/orimmunomodulatory agents, e.g. including corticoids, such asglucocorticoids, and derivatives thereof. However, it is known thatresistance to such agents, e.g. corticoid-resistance, exists or maydevelop, which means that such agents are then not active, or notsufficiently active, for the successful treatment of a variety ofdiseases.

Surprisingly, we have now found that resistance to such agents may beovercome, if a combination treatment is applied.

In one aspect the present invention provides the use of a combination ofa corticoid and a compound of formula

wherein either

R₁ is a group (a) of formula

wherein

R₅ is chloro, bromo, iodo or azido,

R₆ is hydroxy or methoxy, and

R₄ is hydroxy and there is a single bond in 10,11 position; or absent,and there is a double bond in 10,11 position or

R₁ is a group (b) or (c) of formula

wherein

R₆ is as defined above, and

R₄ is hydroxy and there is a single bond in 10,11 position,

R₂ is oxo and there is a single bond in 23,24 position; hydroxy andthere is a single or double bond in 23,24 position; or absent and thereis a double bond in 23,24 position;

R₃ is methyl, ethyl, propyl or allyl,

for the manufacture of a medicament for the treatment of acorticoid-resistant disease and/or a calcineurin inhibitor-resistantdisease, e.g. for the treatment of a disease wherein a compound offormula I alone or a corticoid alone is ineffective or insufficientlyeffective.

Therapeutically ineffective or insufficiently effective means that acompound of a combination of the present invention alone does not showefficacy or does not show sufficient efficacy in a clinical environment.We have established in vitro systems, e.g. as described herein, in whicha corticoid alone or a calcineurin inhibitor alone, under certainconditions, does not inhibit T-cell proliferation to a degree necessaryfor therapeutic treatment. These systems thus serve as an in vitroexperimental models of corticoid and/or calcineurin inhibitorresistance.

We have defined that a compound of the combination of the presentinvention alone with an inhibitory effect of less than 35%, e.g. lessthan 25%, such as less than 20%, is insufficient for therapeutictreatment. On the other hand, we have defined that a compound of acombination of the present invention alone is sufficient for therapeutictreatment if an inhibitory effect of at least 60% and more, such as 80%,90%, up to practically 100% is achieved.

Compounds of formula I are e.g. disclosed in EP-B-0427680, wherein in acompound of formula I preferred substituents are indicated, whichpreferred substituents are also preferred substituents in the presentapplication; e.g. in a compound of formula I each single definedsubstituent may be a preferred substituent, e.g. independently of eachother substituent defined; e.g. including a compound of formula I_(P),such as disclosed in EP-B-0427680 in Example 66a as “33-epi-33-chloro-FR520”, also known as “ASM981”.

In another aspect the present invention provides the use of acombination of a corticoid and a compound of formula I according to thepresent invention, wherein a compound of formula I is a compound offormula

A corticoid in a combination of the present invention includespharmaceutically active corticoids and derivatives thereof, e.g.including corticosteroids, such as glucocorticoids (i.e. havingglucocorticoid-like activity), e.g. which show pharmaceutical activity,as well as nonsteroidal ligands of the glucocorticoid receptor,

e.g. including corticoids in free form and in the form of

-   -   esters, e.g. including mono- and diesters, e.g. in the form of        salts, e.g. sodium,    -   acetals and ketals, such as acetonides,

e.g. in the form of salts and solvates, where applicable.

Examples include alclomethasone, (e.g. -diproprionate), amicinonide,beclomethasone (e.g. -dipropionate), betamethasone (e.g. -acetate,-benzoate, -dipropionate, sodium phosphate, -valerate), budesonide,carbenoxolone (e.g. -sodium), ciclesonide, clobetasole (e.g.propionate), clobetasone (e.g. butyrate), clocortolone (e.g. -acetate,-pivalate), cloprednol, corticosterone, corticotropin (e.g. -zinchydroxide), cortisol, cortisone (e.g. -acetate), cortivazol,deflazacort, descinolone (e.g. -acetonide), desonide, dexamethasone(e.g. sodium phosphate, -acetate, -isomicotinate), desoxymethasone,diflorasone (e.g. diacetate), difluocortolone (e.g. -pivalate,valerate), difluprednate, flucloronide, fludrocortisone,fludroxycortide, flumethasone (e.g. -pivalate), flunisolide, fluocortin(butyl), fluocinonide, fluocinolone (e.g. -acetonide), fluocortolone(e.g. -caproate), fluorometholone, fluperolone (e.g. -acetate),fluprednidene (e.g. -21-acetal, -acetate), fluprednisolone (e.g.-valerate), flurandrenolide, fluticasone (e.g. -propionate, valerate),formocortal, halcinonide, halobetasol (e.g. -propionate), halomethasone(e.g. monohydrate), hydrocortisone (e.g. -acetate, -buteprat, -butyrate,cypionate, -sodium phosphate, -sodium succinate, -hemisuccinate,-valerate), medrysone, methylprednisolone (e.g. -acetate, -sodiumphosphate, -sodium succinate, aceponate), momethasone (e.g. fuorate),nivazol, paramethasone (e.g. -acetate), prednicarbate, prednisolone(e.g. including -acetate, -hemisuccinate, -sodium phosphate, -sodiumsuccinate, -tebutate), prednisone, prednisolone, prednival,prednylidene, rofleponide (e.g. palmitate), ticabesone (e.g.-propionate), tipredane, tralonide, triamcinolone (e.g. -acetonide,-acetonide sodium phosphate, -diacetate), e.g., and pharmacodynamicequivalents thereof, preferably hydrocortisone, betamethasone, e.g.betamethasone 17-valerate, or dexamethasone.

Pharmacodynamic equivalents are meant to include corticoids, havingsimilar pharmaceutical activity in comparison with specific corticoidslisted herein.

Pharmaceutical excipient includes e.g. appropriate carrier and/ordiluent, e.g. including fillers, binders, disintegrators, flowconditioners, lubricants, sugars and sweeteners, fragrances,preservatives, stabilizers, wetting agents and/or emulsifiers,solubilizers, salts for regulating osmotic pressure and/or buffers.

In another aspect the present invention provides the use of acombination of a corticoid and a compound of formula I according to thepresent invention, wherein the corticoid is selected from the groupconsisting of hydrocortisone, betamethasone, e.g. betamethasone17-valerate, and dexamethasone.

A compound of a combination according to the present invention may be infree form, in the form of a salt, in solvate form or in the form of asalt and a solvate, where salts and/or solvates exist.

In another aspect the present invention provides

-   -   the use of a combination of a corticoid and a compound of        formula I according to the present invention, wherein a compound        of formula I is in the form of a salt;    -   the use of a combination of a corticoid and a compound of        formula I according to the present invention, wherein the        corticoid is in the form of a salt;    -   the use of a combination of a corticoid and a compound of        formula I according to the present invention, wherein a compound        of formula I and a corticoid both are in the form of a salt.

Corticoid-resistant diseases are known and include, e.g.

-   -   alopecia, e.g. alopecia totalis or alopecia universalis,    -   allergies, e.g. contact allergies,    -   amyloidosis, e.g. systemic amyloidosis,    -   arteritis, e.g. Takayasu's arteritis,    -   arthritis, e.g. (juvenile) rheumatoid arthritis, juvenile        oligoarthritis, sarcoidosis arthritis,    -   arthropathy, e.g. spondyloarthropathy,    -   asthma, e.g. bronchial asthma, chronic asthma,    -   colitis,    -   conjunctivitis, e.g. keratoconjunctivitis,    -   Crohn's disease, including corticosteroid-resistant pyoderma        gangrenosum associated with Crohn's disease, refractory Crohn's        disease,    -   cystic fibrosis,    -   dermatitis (=dermatosis), such as contact, atopic, allergic        contact and solar dermatitis, lichenoid dermatitis, ulcerative        dermatitis,    -   Multiple Sclerosis, e.g. Encephalo Multiple Sclerosis,    -   eczema,    -   Graves diseases, e.g. Graves' ophthalmopathy,    -   Graft Versus Host Disease (GVHD),    -   hemangiomas,    -   Hepatitis,    -   Inflammatory Bowel Disease (IBD),    -   insulin-dependent diabetes,    -   intraocular inflammatory diseases,    -   keratitis,    -   Macrophage activation syndrome,    -   myasthenia, e.g. myasthenia gravis,    -   myelitis, e.g. encephalomyelitis,    -   myositis, e.g. (juvenile) dermatomyositis (DM), polymyositis        (PM) and inclusion body myositis (IBM),    -   nephritis, e.g. glomerulonephritis, nephritic syndrome,    -   ophthalmia, e.g. sympathetic ophthalmia,    -   pneumonitis,    -   polyarthritis, e.g. chronic polyarthrits such as Still's        disease,    -   psoriasis,    -   pulmonary or lung diseases, e.g. chronic obstructive pulmonary        disease (COPD),    -   retinal detachment    -   sarcoidosis or neurosarcoidosis,    -   scleritis,    -   sclerosis, e.g. glomerulosclerosis,    -   septic shock,    -   Sjogren's syndrome,    -   systemic lupus erythematosus,    -   transplant rejection, e.g. (renal or kidney) allograft        rejection,    -   thrombocytopenic purpura, e.g. immune thrombocytopenic purpura        (ITP), (chronic) idiopathic thrombocytopenic purpura,    -   ulcerative colitis,    -   urticaria, e.g. (chronic) idiopathic urticaria, urticarial        vasculitis,    -   uveitis, such as anterior uveitis, chronic uveitis, peripheral        uveitis, refractory uveitis, Behcet's uveitis, granulomatous        uveitis such as Vogt-Koyanagi-Harada disease.

In another aspect the present invention provides the use of acombination of a corticoid and a compound of formula I, e.g. a compoundof formula I_(p), according to the present invention, wherein thedisease is a disease in which T cells (i.e. T lymphocytes) are involvedin the pathophysiology of the disease, such as T-cell mediated acute orchronic inflammatory diseases or disorders or autoimmune diseases,

e.g. a disease selected from the group consisting of

-   -   Graft Versus Host Disease (GVHD),    -   autoimmune diseases and inflammatory conditions, in particular        inflammatory conditions with an etiology including an autoimmune        component, such as hematological disorders, including e.g.        hemolytic anemia, aplastic anemia, pure red cell anemia and        idiopathic thrombocytopenia, rheumatoid arthritis, systemic        Lupus erythematosus, polychondritis, scleroderma, Wegener's        granulomatosis, chronic active hepatitis, Hashimoto's        thyroiditis, Steven-Johnson syndrome, idiopathic sprue,        autoimmune inflammatory bowel disease, Graves disease,        sarcoidosis, multiple sclerosis, interstitial lung fibrosis,        Myasthenia gravis, glomerulonephritis (with and without        nephritic syndrome), juvenile dermatomyositis, juvenile diabetes        (diabetes mellitus type I), immune-mediated conditions of the        eye, e.g. uveitis (anterior and posterior), keratoplasty,        chronic keratitis, keratoconjunctivitis sicca, vernal        keratoconjunctivitis;    -   cutaneous manifestations of immunologically-mediated illnesses;    -   inflammatory and hyperproliferative skin diseases, such as        psoriasis, atopic dermatitis, contact dermatitis and further        eczematous dermatitides, seborrheic dermatitis, Lichen planus,        Pemphigus, bullous Pemphigoid, Epidermolysis bullosa, urticaria,        angioedemas, vasculitides, erythemas, cutaneous eosinophilias,        Lupus erythematosus and acne;    -   allergic conditions, e.g. vernal conjunctivitis, ocular allergy;    -   inflammatory nervous injury, e.g. brain inflammation;    -   cerebral anoxia, hypoxia or ischemia;    -   asthma,    -   chronic obstructive pulmonary disease (COPD),    -   inflammatory bowel disease (IBD), including ulcerative colitis        and Crohn's disease;    -   multi-drug resistance (MDR), and    -   Alopecia areata,

wherein a compound of formula I, e.g. a compound of formula I_(p), aloneor a corticoid alone is ineffective or insufficiently effective.

In another aspect the present invention provides the use of acombination of a corticoid and a compound of formula I according to thepresent invention, wherein the disease is selected from the groupconsisting of atopic dermatitis, psoriasis, psoriatic arthritis,rheumatoid arthritis, asthma, ulcerative colitis and Crohn's disease.

Treatment includes treatment and prophylaxis.

For such treatment, the appropriate dosage will, of course, varydepending upon, for example, the chemical nature and the pharmacokineticdata of a compound of the present invention employed, the individualhost, the mode of administration and the nature and severity of theconditions being treated. However, in general, for satisfactory resultsin larger mammals, for example humans, a calcineurin inhibitor, e.g.pimecrolimus, may be provided as a solution or cream in the range fromabout 0.1% to 5% w/v or w/w when administered locally, wherein thedosage will depend on the kind of disease to be treated as well as onthe administration site, or in the range of 10 mg to 120 mg per patient,e.g. 0.1 mg/kg to 2 mg/kg, of a calcineurin inhibitor, e.g.pimecrolimus, when administered systemically, e.g. orally, and thecorticoid is given in dosages as known for standard therapies, such ase.g. in a range of 0.5 to 5% in case of topical application or in arange of 0.25 to 2500 mg, preferably 1 to 500 mg, such as 1 to 50 mg,when administered systemically, e.g. orally.

In another aspect the present invention provides the use of acombination of a corticoid and a calcineurin inhibitor for themanufacture of a medicament, e.g. a pharmaceutical composition, for thetreatment of a corticoid-resistant disease and/or a calcineurininhibitor-resistant disease wherein T cells are involved in thepathophysiology of the disease, with the proviso that focal segmentalglomerulosclerosis wherein T cells are involved in the pathophysiologyare excluded, e.g. for the treatment of diseases wherein a calcineurininhibitor or a corticoid alone is ineffective or insufficientlyeffective.

In another aspect the present invention provides the use of acombination of a calcineurin inhibitor and a corticoid according to thepresent invention, wherein the disease is selected from the groupconsisting of

-   -   Graft-Versus-Host Diseases (GVHD),    -   autoimmune diseases and inflammatory conditions, in particular        inflammatory conditions with an etiology including an autoimmune        component, such as hematological disorders, including e.g.        hemolytic anemia, aplastic anemia, pure red cell anemia and        idiopathic thrombocytopenia, rheumatoid arthritis, systemic        Lupus erythematosus, polychondritis, scleroderma, Wegener's        granulomatosis, chronic active hepatitis, Hashimoto's        thyroiditis, Steven-Johnson syndrome, idiopathic sprue,        autoimmune inflammatory bowel disease, Graves disease,        sarcoidosis, multiple sclerosis, interstitial lung fibrosis,        Myasthenia gravis, glomerulonephritis (with and without        nephritic syndrome), juvenile dermatomyositis, juvenile diabetes        (diabetes mellitus type I), immune-mediated conditions of the        eye, e.g. uveitis (anterior and posterior), keratoplasty,        chronic keratits, keratoconjunctivitis sicca, vernal        keratoconjunctivitis; cutaneous manifestations of        immunologically-mediated illnesses;    -   inflammatory and hyperproliferative skin diseases, such as        psoriasis, atopic dermatitis, contact dermatitis and further        eczematous dermatitides, seborrheic dermatitis, Lichen planus,        Pemphigus, bullous Pemphigoid, Epidermolysis bullosa, urticaria,        angioedemas, vasculitides, erythemas, cutaneous eosinophilias,        Lupus erythematosus and acne;    -   allergic conditions, e.g. vernal conjunctivitis, ocular allergy;    -   inflammatory nervous injury, e.g. brain inflammation;    -   cerebral anoxia, hypoxia or ischemia;    -   asthma,    -   inflammatory bowel disease (IBD), including ulcerative colitis        and Crohn's disease;    -   multi-drug resistance (MDR),    -   ulcers, e.g. gastric ulcers    -   vascular damage caused by ischemic diseases and thrombosis    -   necrotizing lesions associated with thermal burns and    -   Alopecia areata,

wherein a calcineurin inhibitor alone or a corticoid alone isineffective or insufficiently effective.

“Corticoid-resistant disease” and/or “calcineurin inhibitor-resistantdisease” are as defined above.

Calcineurin is a calcium/calmodulin-regulated protein phosphataseinvolved in intracellular signalling. For reviews on calcineurin, seee.g. Rusnak and Mertz, Physiol. Rev. 80, 1483-1521 (2000) and Feske etal., Biochem. Biophys. Commun. 311, 1117-1132 (2003). Calcineurininhibitors are substances which block calcineurin dephosphorylation ofappropriate substrates.

A calcineurin inhibitor of the present invention is preferably animmunophilin binding compound having calcineurin inhibitory activity.

Immunophilin binding calcineurin inhibitors are compounds formingcalcineurin inhibiting complexes with immunophilins, e.g. cyclophilinand macrophilin.

Examples of cyclophilin-binding calcineurin inhibitors are cyclosporinsor cyclosporin derivatives (hereinafter cyclosporins) and examples ofmacrophilin-binding calcineurin inhibitors are ascomycin and ascomycinderivatives (hereinafter ascomycins), see e.g. Liu et al., Cell 66,807-815 (1991) and Dumont et al., J. Exp. Med., 176, 751-780 (1992), aswell as tacrolimus (FK506).

Cyclosporins and their preparation are e.g. disclosed in U.S. Pat. No.4,117,118, wherein in a compound of formula I preferred substituents areindicated, which preferred substituents are also preferred substituentsin the present application; e.g. in a compound of formula I each singledefined substituent may be a preferred substituent, e.g. independentlyof each other substituent defined. Cyclosporin, originally extractedfrom the soil fungus Potypaciadium infilatum, has a cyclic 11-amino acidstructure and includes e.g. Cyclosporins A through I, such asCyclosporin A, B, C, D and G, preferably Cyclosporin A.

Ascomycins and their preparation are known. Ascomycin (FR 520) is amacrolide antibiotic disclosed e.g. in U.S. Pat. No. 3,244,592 and in EP349061, wherein in a compound of formula I preferred substituents areindicated, which preferred substituents are also preferred substituentsin the present application; e.g. in a compound of formula I each singledefined substituent may be a preferred substituent, e.g. independentlyof each other substituent defined. A wide range of ascomycin derivativesare known, which are either naturally occurring amongst fungal speciesor are obtainable by manipulation of fermentation procedures or bychemical derivatization.

Ascomycins include e.g. a compound of formula I, as described above,preferably pimecrolimus.

In another aspect the present invention provides the use of acombination of a corticoid and a calcineurin inhibitor according to thepresent invention, wherein a calcineurin inhibitor is a compound offormula I, wherein the substituents are as described above, preferably acompound of formula I_(P).

In another aspect the present invention provides the use of acombination of a calcineurin inhibitor and a corticoid according to thepresent invention, wherein a calcineurin inhibitor is a compound offormula

wherein

R₁ is hydroxy or protected hydroxy,

R₂ is hydrogen, hydroxyl or protected hydroxyl,

R₃ is methyl, ethyl, propyl or allyl,

n is an integer of 1 or 2, and

the symbol of a line and dotted line is a single bond.

Compounds of formula II are e.g. disclosed in EP-B-0184162, wherein in acompound of formula I preferred substituents are indicated, whichpreferred substituents are also preferred substituents in the presentapplication; e.g. in a compound of formula I of the EP-B-0184162, eachsingle defined substituent may be a preferred substituent, e.g.independently of each other substituent defined. A preferred compound isthe compound FK 506 (tacrolimus) of formula II_(FK).

In another aspect the present invention provides the use of acombination of a calcineurin inhibitor and a corticoid according to thepresent invention, wherein a calcineurin inhibitor is a compound offormula

In another aspect the present invention provides the use of acombination of a calcineurin inhibitor and a corticoid according to thepresent invention, wherein a calcineurin inhibitor is a compound offormula

wherein R is methyl, ethyl, propyl, isopropyl or —CH(OH)CH₃, preferablyR is ethyl (Cyclosporin A).

In another aspect the present invention provides the use of acombination of a corticoid and a calcineurin inhibitor according to thepresent invention, wherein a corticoid is selected from corticoids asdescribed above, preferably the corticoid is selected from the groupconsisting of hydrocortisone, betamethasone, e.g. betamethasone17-valerate, and dexamethasone.

A compound of a combination of a calcineurin inhibitor and a corticoidmay be in free form, in the form of a salt, in solvate form or in theform of a salt and a solvate, where salts and/or solvates exist.

In another aspect the present invention provides

-   -   the use of a combination of a corticoid and a calcineurin        inhibitor according to the present invention, wherein a        calcineurin inhibitor is in the form of a salt;    -   the use of a combination of a corticoid and a calcineurin        inhibitor according to the present invention, wherein the        corticoid is in the form of a salt;    -   the use of a combination of a corticoid and a calcineurin        inhibitor according to the present invention, wherein a        calcineurin inhibitor and a corticoid both are in the form of a        salt.

A combination according to the present invention may contain one or morecalcineurin inhibitors and one or more corticoids, and containspreferably one calcineurin inhibitor and one corticoid.

The compounds of a combination of the present invention may be used,e.g. administered, in free form or in the form of a pharmaceuticallyacceptable salt, e.g. an acid addition salt or metal salt; optionally inthe form of a solvate. Corticoids may additionally be in the form ofesters, acetonides, e.g. and additionally in the form of salts. Thecompounds of a combination of the present invention in the form of asalt/ester/acetonide/solvate/ exhibit the same order of activity as thecompounds used in the present invention in free form; optionally in theform of a solvate.

Treatment and dosage are as described above for a combination of acompound of formula I and a corticoid.

The ratio of calcineurin inhibitor, e.g. including a compound of formulaI, II or III, to corticoid depends on various factors, such as e.g. thepotency of each single compound.

In another aspect the present invention provides a pharmaceuticalcomposition comprising, beside pharmaceutically acceptable excipient, incombination a compound of formula I_(p) and hydrocortisone.

In another aspect the present invention provides a pharmaceuticalcomposition comprising, beside pharmaceutically acceptable excipient, incombination a compound of formula I_(p) and betamethasone, e.g.betamethasone 17-valerate.

In another aspect the present invention provides a pharmaceuticalcomposition comprising, beside pharmaceutically acceptable excipient, incombination a compound of formula I_(p) and dexamethasone.

In a combination according to the present invention, pharmaceuticallyacceptable excipient, such as appropriate carrier and/or diluent, e.g.includes fillers, binders, disintegrators, flow conditioners,lubricants, sugars and sweeteners, fragrances, preservatives,stabilizers, wetting agents and/or emulsifiers, solubilizers, salts forregulating osmotic pressure, buffers.

A combination of the present invention includes

-   -   fixed combinations, in which both pharmaceutically active agents        are in the same formulation;    -   kits, in which both pharmaceutically active agents in separate        formulations are sold in the same package, e.g. with        instructions for co-administration; and    -   free combinations in which the pharmaceutically active agents        are packaged separately, but instructions for simultaneous or        sequential administration are given.

Pharmaceutical compositions of the present invention may be manufacturedaccording, e.g. analogously, to a method as conventional, e.g. bymixing, granulating, coating, dissolving or lyophilizing processes. Unitdosage forms may contain, for example, from about 0.5 mg to about 1000mg, such as 1 mg to about 500 mg of pharmaceutically active compounds. Apharmaceutical composition of and for use according to the presentinvention may be administered by any conventional route, for exampleenterally, e.g. including nasal, buccal, rectal, oral, administration;parenterally, e.g. including intravenous, intramuscular, subcutaneousadministration; or topically; e.g. including epicutaneous, intranasal,intratracheal administration;

e.g. in form of coated or uncoated tablets, capsules, injectablesolutions or suspensions, e.g. in the form of ampoules, vials, in theform of creams, gels, pastes, inhaler powder, foams, tinctures, lipsticks, drops, sprays, or in the form of suppositories.

Calcineurin inhibitors, including e.g. compounds of formulae I, II andIII, and corticoids are known or may be obtained according, e.g.analogously, to a method as conventional.

A combination of the present invention may comprise beside a calcineurininhibitor and a corticoid as active ingredients further pharmaceuticallyactive compounds. Such further pharmaceutically active compounds includeother anti-inflammatory, immunomodulatory and anti-proliferative agents.

In another aspect the present invention provides a pharmaceuticalcomposition of the present invention, further comprising anotherpharmaceutically active agent.

We have established in vitro systems using human peripheral bloodmononuclear cells (PBMC), that serve as in vitro experimental models ofresistance to corticoids and/or calcineurin inhibitors, in which acorticoid alone or a calcineurin inhibitor, including e.g. Cyclosporin Aand a compound of formula I, II or III, alone exert either no effect atall, or only a partial inhibitory effect on T-cell proliferation. Thesesystems which are described herein as corticoid-resistant and/orcalcineurin inhibitor-resistant, e.g. compound of formula I-resistant,employ a high cell density, such as 50,000-200,000 cells/well in a96-well plate and powerful stimuli of T-cell proliferation, namely thesuperantigen Staphylococcal Enterotoxin B (SEB) and/or the combinationof anti-CD3 plus anti-CD28 monoclonal antibodies. This contrasts withsystems employing lower cell densities and/or weaker stimuli, such ase.g. anti-CD3 antibody alone, in which corticoids and calcineurininhibitors exert a complete, or almost complete, inhibition of T-cellproliferation at nanomolar to sub-nanomolar concentrations.

Surprisingly, in the resistant system described herein we have foundthat combinations of a calcineurin inhibitor and a corticoid stronglyinhibit T-cell proliferation (e.g. 60% inhibition up to completeinhibition), whereas the single components of the combination do notinhibit at all or do not inhibit by more than 35%. Determinations andmeasurements are as set out under “Methods” in the Examples.

As shown in the Examples, we have found in our in vitro systems that acompound of formula I, II or III, e.g. I_(P), II_(FK) or Cyclosporin Aalone, or hydrocortisone alone, or betamethasone 17-valerate alone, ordexamethasone alone may show either essentially no inhibition or only apartial inhibition, e.g. less than 35% of T-cell proliferation, whereasa combination of hydrocortisone and a compound of formula I_(P), or acombination of betamethasone 17-valerate and a compound of formulaI_(P), or a combination of dexamethasone and a compound of formulaI_(P), or a combination of hydrocortisone and a compound of formulaII_(FK), or a combination of dexamethasone and a compound of formulaII_(FK), or a combination of hydrocortisone and Cyclosporin A, or acombination of betemethasone 17-valerate and Cyclosporin A, or acombination of dexamethasone and Cyclosporin A in the same assays showsan inhibition of at least 60% up to complete inhibition. We thus havefound that a combination of the compounds of the present invention mayshow activity in cases where the single components do not showinhibition or show inhibition of less than 35%, e.g. less than 25%, suchas less than 20%.

In the following examples temperatures are given in degrees Celsius (°C.) and are uncorrected.

The following abbreviations are used:

-   ASM a compound of formula I_(P) (ASM981, pimecrolimus)-   BETA betamethasone 17-valerate-   BrdU 5-bromo-2-deoxy-uridine-   CsA Cyclosporin A-   DEX dexamethasone-   DMSO dimethylsulfoxide-   FCS fetal calf serum-   FK a compound of formula II_(FK) (FK 506, tacrolimus)-   HC hydrocortisone-   mAb monoclonal antibody-   μg microgram-   μM micromolar-   nM nanomolar-   OD Optical Density-   PBMC peripheral blood mononuclear cells-   rpm revolutions per minute-   RPMI medium developed at Roswell Park Memorial Institute-   RT room temperature-   SD Standard Deviation-   SEB Staphylococcal Enterotoxin B (a superantigen)-   STIM-C stimulated control-   UNSTIM-C unstimulated control

EXAMPLE A

Materials

The mouse anti-CD3 mAb (clone SPV-T3/1, isotype IgG2a), which stimulatesthe human T cell receptor, is known (Spits H., Keizer G., Borst J. etal., (1983) Characterization of monoclonal antibodies against cellsurface molecules associated with cytotoxic activity of natural andactivated killer cells and cloned CTL lines, Hybridoma 2:423-437) andmay be prepared as appropriate. The mouse anti-CD28 mAb (clone CD28.2,isotype IgG1, κ) is obtained from BD Biosciences (Catalog #555725). SEBis obtained from Toxin Technology Inc. (Sarasota, Fla., USA; Catalog#TX-BT202). Cell proliferation ELISA kits (colorimetric) based on themeasurement of BrdU incorporation during DNA synthesis are obtained fromRoche Molecular Biochemicals (Mannheim, Germany). These kits are usedaccording to the manufacturer's instructions.

Cell Cultures

PBMC are isolated from human buffy coats by Ficoll/Hypaquecentrifugation under sterile conditions, frozen in 90% FCS+10% DMSO andstored in liquid nitrogen. For each experiment frozen PBMC are thawedand then washed and resuspended in a culture medium consisting of RPMI1640 (Gibco-BRL, Paisley, UK), supplemented with 10% heat-inactivatedFCS (Gibco), 2 mM glutamine, 0.1 mg/ml streptomycin, and 100 units/mlpenicillin (Gibco). 0.1 ml of cell suspension obtained are added perwell to columns 2-11 of 96-well flat-bottomed cell culture plates (Nunc,Roskilde, Denmark), whereas column 12 contains only medium and serves asthe medium blank. Only the inner wells receive cells and are used in theexperiments (i.e. column 1 and rows A and H are excluded; receiving onlymedium). The solvent control and test compounds (0.05 ml/well added) arefirst incubated with cells at 37° C./5% CO₂ for 2 hours. The stimulantis then added (0.05 ml/well) to all wells to the indicated finalconcentrations, except to the unstimulated control, which is receivingculture medium (0.05 ml/well), and the plates are incubated for another68 hours at 37° C./5% CO₂ followed by the addition of BrdU (0.02 ml/wellof the BrdU labeling solution [=10 μM BrdU in cell culture medium] fromthe ELISA kit). After further incubation at 37° C./5% CO₂ for 4 hours,the plates are centrifuged at 1200 rpm (300×g) for 10 min at RT. Afterremoval of the supernatants, the plates are incubated at 60° C. for 1hour to dry the cells. The solvent control and each compound or compoundcombination (at the indicated final concentrations) are tested intriplicate (i.e. 3 wells per concentration). Test compounds (i.e.compound-combinations of the present invention and single compounds ofsuch combinations) are dissolved in DMSO and then subsequently dilutedinto cell culture medium so that the final DMSO concentration in thecell plates does not exceed 0.2% (v/v).

T-Cell Proliferation Assay

The plates are processed for the determination of cell proliferationbased on the incorporation of BrdU during DNA synthesis using an ELISAkit (Roche Molecular Biochemicals) according to the manufacturer'sinstructions. The optical densities are measured in a microtiter platereader at 450 nm, with a reference wavelength of 690 nm. The absorbancedata are analyzed by the software program Excel™. The average of thevalues in the cell-free wells is used as the blank and subtracted fromthe other values. The averages and standard deviations of theabsorbances for each compound and compound combination are calculatedand then normalized to the solvent control containing stimulus, (i.e.stimulated control), which is defined as 100%. The % inhibition for eachcompound and compound combination is also calculated.

In the following Examples 1 to 27, the T-cell proliferation assay iscarried out analogously as described herein.

On day 0 the cells are seeded into 96-well cell culture plates andpreincubated with compound(s) for 2 hours before addition of stimulus.On day 3 (i.e. 68 hours later) BrdU is added and the plates areincubated for 4 hours before being processed to determine BrdUincorporation via the BrdU ELISA.

In the TABLES, the following terms are defined:

-   -   “Unstimulated Control”: the mean of the OD values measured for        the unstimulated control (i.e. cells minus stimulus and        compound(s)), along with the calculated SD values.    -   “Stimulated Control”: the mean of the OD values measured for the        stimulated control (i.e. cells plus stimulus and minus        compound(s)), along with the calculated SD values.    -   “Compound(s)”: the mean of the OD values measured for the        samples in the presence of stimulus and compound(s), along with        the calculated SD values.

“Normalized OD [or SD] (% Stimulated Control)”: the OD and SD values arenormalized relative to the Stimulated Control, which is set to 100.

“% Inhibition”: defined as 100%−[(Sample OD−Unstimulated ControlOD)/(Stimulated Control OD−Unstimulated Control OD)]×100%. A negativevalue indicates stimulation of proliferation relative to the stimulatedcontrol (defined as 0% inhibition). A value greater than 100 indicatesinhibition to below the level of the unstimulated control (defined as100% inhibition).

EXAMPLE 1

Cells/Well: 200000

Compound(s): HC (10 μM), ASM (30 nM), BETA (300 nM), a combination of HC(10 μM)+ASM (30 nM) and a combination of BETA (300 nM)+ASM (30 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 1 below are obtained. TABLE 1 UNSTIM- STIM-ASM + ASM + C C HC ASM BETA HC BETA OD 0.068 0.768 0.820 0.721 0.8080.300 0.228 SD 0.012 0.028 0.015 0.099 0.034 0.060 0.022 Normalized OD8.9 100 106.8 93.9 105.2 39.1 29.7 (% STIM-C) Normalized SD 1.6 3.6 2.012.9 4.4 7.8 2.9 (% STIM-C) % Inhibition 100 0 −7.4 6.7 −5.7 66.9 77.1

From TABLE 1 it is evident that ASM alone, HC alone and BETA alone showessentially no inhibition of T-cell proliferation in this system,whereas combinations of ASM+HC and ASM+BETA show inhibition of more than65%.

EXAMPLE 2

Cells/Well: 100000

Compound(s): HC (10 μM), ASM (30 nM), BETA (300 nM), a combination of HC(10 μM)+ASM (30 nM) and a combination of BETA (300 nM)+ASM (30 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 2 below are obtained. TABLE 2 UNSTIM-CSTIM-C HC ASM BETA ASM + HC ASM + BETA OD 0.082 0.912 0.839 0.776 0.7960.301 0.189 SD 0.003 0.085 0.055 0.091 0.126 0.015 0.046 Normalized OD9.0 100 92.0 85.1 87.3 33.0 20.7 (% STIM-C) Normalized SD 0.3 9.3 6.010.0 13.8 1.6 5.0 (% STIM-C) % Inhibition 100 0 8.8 16.4 14.0 73.6 87.1

From TABLE 2 it is evident that ASM alone, HC alone and BETA alone showless than 17% inhibition of T-cell proliferation in this system, whereascombinations of ASM+HC and ASM+BETA show inhibitions of more than 73%.

EXAMPLE 3

Cells/Well: 50000

Compound(s): HC (10 μM), ASM (30 nM), BETA (300 nM), a combination of HC(10 μM)+ASM (30 nM) and a combination of BETA (300 nM)+ASM (30 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 3 below are obtained. TABLE 3 UNSTIM-CSTIM-C HC ASM BETA ASM + HC ASM + BETA OD 0.065 0.878 0.957 0.994 0.8730.115 0.102 SD 0.012 0.087 0.053 0.039 0.075 0.017 0.035 Normalized OD7.4 100 109.0 113.2 99.4 13.1 11.6 (% STIM-C) Normalized SD 1.4 9.9 6.04.4 8.5 1.9 4.0 (% STIM-C) % Inhibition 100 0 −9.7 −14.3 0.6 93.8 95.4

From TABLE 3 it is evident that ASM alone, HC alone and BETA alone showessentially no inhibition of T-cell proliferation in this system,whereas combinations of ASM+HC and ASM+BETA show inhibitions of morethan 93%.

EXAMPLE 4

Cells/Well: 200000

Compound(s): HC (10 μM), ASM (30 nM), BETA (300 nM), a combination of HC(10 μM)+ASM (30 nM) and a combination of BETA (300 nM)+ASM (30 nM)

Stimulus: SEB (0.1 μg/ml)

Results: as set out in TABLE 4 below are obtained. TABLE 4 UNSTIM-CSTIM-C HC ASM BETA ASM + HC ASM + BETA OD 0.069 1.059 0.942 1.015 0.9000.070 0.073 SD 0.006 0.204 0.078 0.065 0.095 0.012 0.010 Normalized OD6.5 100 89.0 95.8 85.0 6.6 6.9 (% STIM-C) Normalized SD 0.6 19.3 7.4 6.19.0 1.1 0.9 (% STIM-C) % Inhibition 100 0 11.8 4.4 16.1 99.9 99.6

From TABLE 4 it is evident that ASM alone, HC alone and BETA alone showless than 17% inhibition of T-cell proliferation in this system, whereascombinations of ASM+HC and ASM+BETA show inhibitions of more than 99%(i.e. essentially complete inhibition).

EXAMPLE 5

Cells/Well: 100000

Compound(s): HC (10 μM), ASM (30 nM), BETA (300 nM), a combination of HC(10 μM)+ASM (30 nM) and a combination of BETA (300 nM)+ASM (30 nM)

Stimulus: SEB (0.1 μg/ml)

Results: as set out in TABLE 5 below are obtained. TABLE 5 UNSTIM-CSTIM-C HC ASM BETA ASM + HC ASM + BETA OD 0.050 0.851 0.855 0.665 1.0010.051 0.043 SD 0.022 0.057 0.100 0.196 0.145 0.017 0.001 Normalized OD5.9 100 100.5 78.1 117.6 6.0 5.1 (% STIM-C) Normalized SD 2.6 6.7 11.823.0 17.0 2.0 0.1 (% STIM-C) % Inhibition 100 0 −0.5 23.2 −18.7 99.9100.9

From TABLE 5 it is evident that ASM alone, HC alone and BETA alone showless than 24% inhibition of T-cell proliferation in this system, whereascombinations of ASM+HC and ASM+BETA show inhibitions of more than 99%(i.e. essentially complete inhibition).

EXAMPLE 6

Cells/Well: 200000

Compound(s): DEX (300 nM), ASM (30 nM), BETA (300 nM), a combination ofDEX (300 nM)+ASM (30 nM) and a combination of BETA (300 nM)+ASM (30 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 6 below are obtained. TABLE 6 UNSTIM-CSTIM-C DEX ASM BETA ASM + DEX ASM + BETA OD 0.022 0.788 0.687 0.7720.703 0.145 0.179 SD 0.003 0.048 0.026 0.152 0.027 0.026 0.035Normalized OD 2.8 100 87.2 98.0 89.2 18.4 22.7 (% STIM-C) Normalized SD0.4 6.1 3.3 19.3 3.4 3.3 4.4 (% STIM-C) % Inhibition 100 0 13.2 2.1 11.183.9 79.5

From TABLE 6 it is evident that ASM alone, DEX alone and BETA alone showless than 14% inhibition of T-cell proliferation in this system, whereascombinations of ASM+DEX and ASM+BETA show inhibitons of more than 79%.

EXAMPLE 7

Cells/Well: 100000

Compound(s): DEX (300 nM), ASM (30 nM), BETA (300 nM), a combination ofDEX (300 nM)+ASM (30 nM) and a combination of BETA (300 nM)+ASM (30 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 7 below are obtained. TABLE 7 UNSTIM-CSTIM-C DEX ASM BETA ASM + DEX ASM + BETA OD 0.024 0.840 0.643 0.7340.692 0.134 0.084 SD 0.009 0.052 0.020 0.051 0.118 0.071 0.011Normalized OD 2.9 100 76.5 87.4 82.4 16.0 10.0 (% STIM-C) Normalized SD1.1 6.2 2.4 6.1 14.0 8.5 1.3 (% STIM-C) % Inhibition 100 0 24.1 13.018.1 86.5 92.6

From TABLE 7 it is evident that ASM alone, DEX alone and BETA alone showless than 25% inhibition of T-cell proliferation in this system, whereascombinations of ASM+DEX and ASM+BETA show inhibitions of more than 86%.

EXAMPLE 8

Cells/Well: 50000

Compound(s): DEX (300 nM), ASM (30 nM), BETA (300 nM), a combination ofDEX (300 nM)+ASM (30 nM) and a combination of BETA (300 nM)+ASM (30 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 8 below are obtained. TABLE 8 UNSTIM-CSTIM-C DEX ASM BETA ASM + DEX ASM + BETA OD 0.023 1.053 0.866 0.9080.794 0.056 0.057 SD 0.004 0.133 0.083 0.146 0.136 0.018 0.003Normalized OD 2.2 100 82.2 86.2 75.4 5.3 5.4 (% STIM-C) Normalized SD0.4 12.6 7.9 13.9 12.9 1.7 0.3 (% STIM-C) % Inhibition 100 0 18.2 14.125.1 96.8 96.7

From TABLE 8 it is evident that ASM alone, DEX alone and BETA alone showless than 26% inhibition of T-cell proliferation in this system, whereascombinations of ASM+DEX and ASM+BETA show inhibitions of more than 96%.

EXAMPLE 9

Cells/Well: 200000

Compound(s): DEX (300 nM), ASM (30 nM), BETA (300 nM), a combination ofDEX (300 nM)+ASM (30 nM) and a combination of BETA (300 nM)+ASM (30 nM)

Stimulus: SEB (0.1 μg/ml)

Results: as set out in TABLE 9 below are obtained. TABLE 9 UNSTIM-CSTIM-C DEX ASM BETA ASM + DEX ASM + BETA OD 0.065 0.905 0.919 0.7520.902 0.052 0.059 SD 0.012 0.070 0.008 0.057 0.130 0.010 0.004Normalized OD 7.2 100 101.5 83.1 99.7 5.7 6.5 (% STIM-C) Normalized SD1.3 7.7 0.9 6.3 14.4 1.1 0.4 (% STIM-C) % Inhibition 100 0 −1.7 18.2 0.4101.5 100.7

From TABLE 9 it is evident that ASM alone, DEX alone and BETA alone showless than 19% inhibition of T-cell proliferation in this system, whereascombinations of ASM+DEX and ASM+BETA show inhibitions of more than 100%(i.e. essentially complete inhibition).

EXAMPLE 10

Cells/Well: 100000

Compound(s): DEX (300 nM), ASM (30 nM), BETA (300 nM), a combination ofDEX (300 nM)+ASM (30 nM) and a combination of BETA (300 nM)+ASM (30 nM)

Stimulus: SEB (0.1 μg/ml)

Results: as set out in TABLE 10 below are obtained. TABLE 10 UNSTIM-CSTIM-C DEX ASM BETA ASM + DEX ASM + BETA OD 0.022 0.980 0.847 0.6600.955 0.023 0.029 SD 0.008 0.153 0.084 0.138 0.053 0.008 0.005Normalized OD 2.2 100 86.4 67.3 97.4 2.3 3.0 (% STIM-C) Normalized SD0.8 15.6 8.6 14.1 5.4 0.8 0.5 (% STIM-C) % Inhibition 100 0 13.9 33.42.6 99.9 99.3

From TABLE 10 it is evident that ASM alone, DEX alone and BETA aloneshow less than 34% inhibition of T-cell proliferation in this system,whereas combinations of ASM+DEX and ASM+BETA show inhibitions of morethan 99% (i.e. essentially complete inhibition).

EXAMPLE 11

Cells/Well: 100000

Compound(s): DEX (300 nM), ASM (30 nM), and a combination of DEX (300nM)+ASM (30 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 11 below are obtained. TABLE 11 ASM +UNSTIM-C STIM-C DEX ASM DEX OD 0.033 1.160 0.885 0.918 0.069 SD 0.0080.060 0.048 0.031 0.007 Normalized OD 2.8 100 76.3 79.1 5.9 (% STIM-C)Normalized SD 0.7 5.2 4.1 2.7 0.6 (% STIM-C) % Inhibition 100 0 24.421.5 96.8

From TABLE 11 it is evident that ASM alone and DEX alone show less than25% inhibition of T-cell proliferation in this system, whereas acombination of ASM+DEX shows an inhibition of more than 96%.

EXAMPLE 12

Cells/Well: 100000

Compound(s): DEX (300 nM), ASM (30 nM), and a combination of DEX (300nM)+ASM (30 nM)

Stimulus: SEB (0.1 μg/ml)

Results: as set out in TABLE 12 below are obtained. TABLE 12 ASM +UNSTIM-C STIM-C DEX ASM DEX OD 0.044 1.307 1.382 1.013 0.022 SD 0.0030.183 0.050 0.135 0.003 Normalized OD 3.4 100 105.7 77.5 1.7 (% STIM-C)Normalized SD 0.2 14.0 3.8 10.3 0.2 (% STIM-C) % Inhibition 100 0 −5.923.3 101.7

From TABLE 12 it is evident that ASM alone and DEX alone show less than24% inhibition of T-cell proliferation in this system, whereas acombination of ASM+DEX shows an inhibition of more than 101% (i.e.essentially complete inhibition).

EXAMPLE 13

Cells/Well: 200000

Compound(s): HC (10 μM), FK (30 nM), BETA (300 nM), a combination of HC(10 μM)+FK (30 nM) and a combination of BETA (300 nM)+FK (30 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 13 below are obtained. TABLE 13 FK + FK +UNSTIM-C STIM-C HC FK BETA HC BETA OD 0.068 0.768 0.820 0.921 0.8080.345 0.152 SD 0.012 0.028 0.015 0.063 0.034 0.155 0.054 Normalized OD8.9 100 106.8 119.9 105.2 44.9 19.8 (% STIM-C) Normalized SD 1.6 3.6 2.08.2 4.4 20.2 7.0 (% STIM-C) % Inhibition 100 0 −7.4 −21.9 −5.7 60.4 88.0

From TABLE 13 it is evident that FK alone, HC alone and BETA alone showessentially no inhibition of T-cell proliferation in this system,whereas combinations of FK+HC and FK+BETA show inhibition of more than60%.

EXAMPLE 14

Cells/Well: 100000

Compound(s): HC (10 μM), FK (30 nM), BETA (300 nM), a combination of HC(10 μM)+FK (30 nM) and a combination of BETA (300 nM)+FK (30 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml) Results: asset out in TABLE 14 below are obtained. TABLE 14 UNSTIM-C STIM-C HC FKBETA FK + HC FK + BETA OD 0.082 0.912 0.839 0.662 0.796 0.243 0.101 SD0.003 0.085 0.055 0.147 0.126 0.046 0.032 Normalized OD 9.0 100 92.072.6 87.3 26.6 11.1 (% STIM-C) Normalized SD 0.3 9.3 6.0 16.1 13.8 5.03.5 (% STIM-C) % Inhibition 100 0 8.8 30.1 14.0 80.6 97.7

From TABLE 14 it is evident that FK alone, HC alone and BETA alone showless than 31% inhibition of T-cell proliferation in this system, whereascombinations of FK+HC and FK+BETA show inhibitions of more than 80%.

EXAMPLE 15

Cells/Well: 200000

Compound(s): DEX (300 nM), FK (30 nM), BETA (300 nM), a combination ofDEX (300 nM)+FK (30 nM) and a combination of BETA (300 nM)+FK (30 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 15 below are obtained. TABLE 15 UNSTIM-CSTIM-C DEX FK BETA FK + DEX FK + BETA OD 0.022 0.788 0.687 0.795 0.7030.081 0.107 SD 0.003 0.048 0.026 0.028 0.027 0.007 0.022 Normalized OD2.8 100 87.2 100.9 89.2 10.3 13.6 (% STIM-C) Normalized SD 0.4 6.1 3.33.6 3.4 0.9 2.8 (% STIM-C) % Inhibition 100 0 13.2 −0.9 11.1 92.3 88.9

From TABLE 15 it is evident that FK alone, DEX alone and BETA alone showless than 14% inhibition of T-cell proliferation in this system, whereascombinations of FK+DEX and FK+BETA show inhibitions of more than 88%.

EXAMPLE 16

Cells/Well: 100000

Compound(s): DEX (300 nM), FK (30 nM), BETA (300 nM), a combination ofDEX (300 nM)+FK (30 nM) and a combination of BETA (300 nM)+FK (30 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 16 below are obtained. TABLE 16 UNSTIM-CSTIM-C DEX FK BETA FK + DEX FK + BETA OD 0.024 0.840 0.643 0.809 0.6920.072 0.086 SD 0.009 0.052 0.020 0.034 0.118 0.024 0.011 Normalized OD2.9 100 76.5 96.3 82.4 8.6 10.2 (% STIM-C) Normalized SD 1.1 6.2 2.4 4.014.0 2.9 1.3 (% STIM-C) % Inhibition 100 0 24.1 3.8 18.1 94.1 92.4

From TABLE 16 it is evident that FK alone, DEX alone and BETA alone showless than 25% inhibition of T-cell proliferation in this system, whereascombinations of FK+DEX and FK+BETA show inhibitions of more than 92%.

EXAMPLE 17

Cells/Well: 50000

Compound(s): DEX (300 nM), FK (30 nM), BETA (300 nM), a combination ofDEX (300 nM)+FK (30 nM) and a combination of BETA (300 nM)+FK (30 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 17 below are obtained. TABLE 17 UNSTIM-CSTIM-C DEX FK BETA FK + DEX FK + BETA OD 0.023 1.053 0.866 0.838 0.7940.030 0.032 SD 0.004 0.133 0.083 0.057 0.136 0.003 0.013 Normalized OD2.2 100 82.2 79.6 75.4 2.8 3.0 (% STIM-C) Normalized SD 0.4 12.6 7.9 5.412.9 0.3 1.2 (% STIM-C) % Inhibition 100 0 18.2 20.9 25.1 99.3 99.1

From TABLE 17 it is evident that FK alone, DEX alone and BETA alone showless than 26% inhibition of T-cell proliferation in this system, whereascombinations of FK+DEX and FK+BETA show inhibitions of more than 99%(i.e. essentially complete inhibition).

EXAMPLE 18

Cells/Well: 100000

Compound(s): DEX (300 nM), FK (30 nM), and a combination of DEX (300nM)+FK (30 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml) Results: asset out in TABLE 18 below are obtained. TABLE 18 FK + UNSTIM-C STIM-CDEX FK DEX OD 0.033 1.160 0.885 0.905 0.048 SD 0.008 0.060 0.048 0.1010.006 Normalized OD 2.8 100 76.3 78.0 4.1 (% STIM-C) Normalized SD 0.75.2 4.1 8.7 0.5 (% STIM-C) % Inhibition 100 0 24.4 22.6 98.7

From TABLE 18 it is evident that FK alone and DEX alone show less than25% inhibition of T-cell proliferation in this system, whereas acombination of FK+DEX shows an inhibition of more than 98% (i.e.essentially complete inhibition).

EXAMPLE 19

Cells/Well: 200000

Compound(s): HC (10 μM), CsA (300 nM), BETA (300 nM), a combination ofHC (10 μM)+CsA (30 nM) and a combination of BETA (300 nM)+CsA (300 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 19 below are obtained. TABLE 19 UNSTIM-CSTIM-C HC CsA BETA CsA + HC CsA + BETA OD 0.068 0.768 0.820 0.760 0.8080.239 0.154 SD 0.012 0.028 0.015 0.048 0.034 0.025 0.045 Normalized OD8.9 100 106.8 99.0 105.2 31.1 20.1 (% STIM-C) Normalized SD 1.6 3.6 2.06.3 4.4 3.3 5.9 (% STIM-C) % Inhibition 100 0 −7.4 1.1 −5.7 75.6 87.7

From TABLE 19 it is evident that CsA alone, HC alone and BETA alone showessentially no inhibition of T-cell proliferation in this system,whereas combinations of CsA+HC and CsA+BETA show inhibition of more than75%.

EXAMPLE 20

Cells/Well: 100000

Compound(s): HC (10 μM), CsA (300 nM), BETA (300 nM), a combination ofHC (10 μM)+CsA (30 nM) and a combination of BETA (300 nM)+CsA (300 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 20 below are obtained. TABLE 20 UNSTIM-CSTIM-C HC CsA BETA CsA + HC CsA + BETA OD 0.082 0.912 0.839 0.783 0.7960.167 0.091 SD 0.003 0.085 0.055 0.119 0.126 0.039 0.015 Normalized OD9.0 100 92.0 85.9 87.3 18.3 10.0 (% STIM-C) Normalized SD 0.3 9.3 6.013.0 13.8 4.3 1.6 (% STIM-C) % Inhibition 100 0 8.8 15.5 14.0 89.8 98.9

From TABLE 20 it is evident that CsA alone, HC alone and BETA alone showless than 16% inhibition of T-cell proliferation in this system, whereascombinations of CsA+HC and CsA+BETA show inhibitions of more than 89%.

EXAMPLE 21

Cells/Well: 50000

Compound(s): HC (10 μM), CsA (300 nM), BETA (300 nM), a combination ofHC (10 μM)+CsA (30 nM) and a combination of BETA (300 nM)+CsA (300 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 21 below are obtained. TABLE 21 UNSTIM-CSTIM-C HC CsA BETA CsA + HC CsA + BETA OD 0.065 0.878 0.957 0.865 0.8730.082 0.055 SD 0.012 0.087 0.053 0.039 0.075 0.024 0.014 Normalized OD7.4 100 109.0 98.5 99.4 9.3 6.3 (% STIM-C) Normalized SD 1.4 9.9 6.0 4.48.5 2.7 1.6 (% STIM-C) % Inhibition 100 0 −9.7 1.6 0.6 97.9 101.2

From TABLE 21 it is evident that CsA alone, HC alone and BETA alone showessentially no inhibition of T-cell proliferation in this system,whereas combinations of CsA+HC and CsA+BETA show inhibitions of morethan 97% (i.e. essentially complete inhibition).

EXAMPLE 22

Cells/Well: 200000

Compound(s): HC (10 μM), CsA (300 nM), BETA (300 nM), a combination ofHC (10 μM)+CsA (30 nM) and a combination of BETA (300 nM)+CsA (300 nM)

Stimulus: SEB (0.1 μg/ml)

Results: as set out in TABLE 22 below are obtained. TABLE 22 UNSTIM-CSTIM-C HC CsA BETA CsA + HC CsA + BETA OD 0.069 1.059 0.942 1.122 0.9000.061 0.074 SD 0.006 0.204 0.078 0.241 0.095 0.005 0.008 Normalized OD6.5 100 89.0 105.9 85.0 5.8 7.0 (% STIM-C) Normalized SD 0.6 19.3 7.422.8 9.0 0.5 0.8 (% STIM-C) % Inhibition 100 0 11.8 −6.4 16.1 100.8 99.5

From TABLE 22 it is evident that CsA alone, HC alone and BETA alone showless than 17% inhibition of T-cell proliferation in this system, whereascombinations of CsA+HC and CsA+BETA show inhibitions of more than 99%(i.e. essentially complete inhibition).

EXAMPLE 23

Cells/Well: 200000

Compound(s): DEX (300 nM), CsA (300 nM), BETA (300 nM), a combination ofDEX (300 nM)+CsA (30 nM) and a combination of BETA (300 nM)+CsA (300 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 23 below are obtained. TABLE 23 UNSTIM-CSTIM-C DEX CsA BETA CsA + DEX CsA + BETA OD 0.022 0.788 0.687 0.7860.703 0.110 0.102 SD 0.003 0.048 0.026 0.088 0.027 0.027 0.028Normalized OD 2.8 100 87.2 99.7 89.2 14.0 12.9 (% STIM-C) Normalized SD0.4 6.1 3.3 11.2 3.4 3.4 3.6 (% STIM-C) % Inhibition 100 0 13.2 0.3 11.188.5 89.6

From TABLE 23 it is evident that CsA alone, DEX alone and BETA aloneshow less than 14% inhibition of T-cell proliferation in this system,whereas combinations of CsA+DEX and CsA+BETA show inhibitions of morethan 88%.

EXAMPLE 24

Cells/Well: 100000

Compound(s): DEX (300 nM), CsA (300 nM), BETA (300 nM), a combination ofDEX (300 nM)+CsA (30 nM) and a combination of BETA (300 nM)+CsA (300 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 24 below are obtained. TABLE 24 UNSTIM-CSTIM-C DEX CsA BETA CsA + DEX CsA + BETA OD 0.024 0.840 0.643 0.8040.692 0.070 0.061 SD 0.009 0.052 0.020 0.022 0.118 0.017 0.006Normalized OD 2.9 100 76.5 95.7 82.4 8.3 7.3 (% STIM-C) Normalized SD1.1 6.2 2.4 2.6 14.0 2.0 0.7 (% STIM-C) % Inhibition 100 0 24.1 4.4 18.194.4 95.5

From TABLE 24 it is evident that CsA alone, DEX alone and BETA aloneshow less than 25% inhibition of T-cell proliferation in this system,whereas combinations of CsA+DEX and CsA+BETA show inhibitions of morethan 94%.

EXAMPLE 25

Cells/Well: 50000

Compound(s): DEX (300 nM), CsA (300 nM), BETA (300 nM), a combination ofDEX (300 nM)+CsA (30 nM) and a combination of BETA (300 nM)+CsA (300 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 25 below are obtained. TABLE 25 UNSTIM-CSTIM-C DEX CsA BETA CsA + DEX CsA + BETA OD 0.023 1.053 0.866 0.8690.794 0.033 0.039 SD 0.004 0.133 0.083 0.026 0.136 0.010 0.013Normalized OD 2.2 100 82.2 82.5 75.4 3.1 3.7 (% STIM-C) Normalized SD0.4 12.6 7.9 2.5 12.9 0.9 1.2 (% STIM-C) % Inhibition 100 0 18.2 17.925.1 99.0 98.4

From TABLE 25 it is evident that CsA alone, DEX alone and BETA aloneshow less than 26% inhibition of T-cell proliferation in this system,whereas combinations of CsA+DEX and CsA+BETA show inhibitions of morethan 98% (i.e. essentially complete inhibition).

EXAMPLE 26

Cells/Well: 200000

Compound(s): DEX (300 nM), CsA (300 nM), BETA (300 nM), a combination ofDEX (300 nM)+CsA (30 nM) and a combination of BETA (300 nM)+CsA (300 nM)

Stimulus: SEB (0.1 μg/ml)

Results: as set out in TABLE 26 below are obtained. TABLE 26 UNSTIM-CSTIM-C DEX CsA BETA CsA + DEX CsA + BETA OD 0.065 0.905 0.919 0.7770.902 0.052 0.064 SD 0.012 0.070 0.008 0.083 0.130 0.025 0.016Normalized OD 7.2 100 101.5 85.9 99.7 5.7 7.1 (% STIM-C) Normalized SD1.3 7.7 0.9 9.2 14.4 2.8 1.8 (% STIM-C) % Inhibition 100 0 −1.7 15.2 0.4101.5 100.1

From TABLE 26 it is evident that CsA alone, DEX alone and BETA aloneshow less than 16% inhibition of T-cell proliferation in this system,whereas combinations of CsA+DEX and CsA+BETA show inhibitions of morethan 100% (i.e. essentially complete inhibition).

EXAMPLE 27

Cells/Well: 100000

Compound(s): DEX (300 nM), CsA (300 nM), and a combination of DEX (300nM)+CsA (300 nM)

Stimulus: anti-CD3 mAb (0.1 μg/ml)+anti-CD28 mAb (1 μg/ml)

Results: as set out in TABLE 27 below are obtained. TABLE 27 UNSTIM-CSTIM-C DEX CsA CsA + DEX OD 0.033 1.160 0.885 0.844 0.048 SD 0.008 0.0600.048 0.014 0.010 Normalized 2.8 100 76.3 72.8 4.1 OD (% STIM-C)Normalized SD 0.7 5.2 4.1 1.2 0.9 (% STIM-C) % Inhibition 100 0 24.428.0 98.7

From TABLE 27 it is evident that CsA alone and DEX alone show less than29% inhibition of T-cell proliferation in this system, whereas acombination of CsA+DEX shows an inhibition of more than 98% (i.e.essentially complete inhibition).

1. Use of a combination of a corticoid and a compound of formula

wherein either R₁ is a group (a) of formula

wherein R₅ is chloro, bromo, iodo or azido, R₆ is hydroxy or methoxy,and R₄ is hydroxy and there is a single bond in 10,11 position; orabsent, and there is a double bond in 10,11 position or R₁ is a groupselected from

wherein R₆ is as defined above, and R₄ is hydroxy and there is a singlebond in 10,11 position, R₂ is oxo and there is a single bond in 23,24position; hydroxy and there is a single or double bond in 23,24position; or absent and there is a double bond in 23,24 position; R₃ ismethyl, ethyl, propyl or allyl, for the manufacture of a medicament forthe treatment of a corticoid-resistant disease and/or a calcineurininhibitor-resistant disease.
 2. Use of claim 1, wherein the compound offormula I is a compound of formula


3. Use of claim 1, wherein the corticoid is selected from the groupconsisting of hydrocortisone, betamethasone and dexamethasone.
 4. Use ofclaim 1, wherein T cells are involved in the pathophysiology of thedisease.
 5. Use of claim 1, wherein the disease is selected from thegroup consisting of atopic dermatitis, psoriasis, psoriatic arthritis,rheumatoid arthritis, asthma, ulcerative colitis and Crohn's disease. 6.Use of a combination of a corticoid and a calcineurin inhibitor for themanufacture of a medicament for the treatment of a corticoid-resistantdisease and/or a calcineurin inhibitor-resistant disease wherein T cellsare involved in the pathophysiology of the disease, with the provisothat focal segmental glomerulosclerosis wherein T cells are involved inthe pathophysiology are excluded.
 7. Use of claim 6, wherein thecalcineurin inhibitor is a compound of formula

wherein substituents are as defined in claim
 1. 8. Use of claim 6,wherein the calcineurin inhibitor is a compound of formula

wherein R₁ is hydroxy or protected hydroxy, R₂ is hydrogen, hydroxyl orprotected hydroxyl, R₃ is methyl, ethyl, propyl or allyl, n is aninteger of 1 or 2, and the symbol of a line and dotted line is a singlebond.
 9. Use of claim 6, wherein the calcineurin inhibitor is a compoundof formula

wherein R is methyl, ethyl, propyl, isopropyl or —CH(OH)CH₃.
 10. Use ofclaim 6, wherein the calcineurin inhibitor is a compound of formulaI_(p) of claim
 2. 11. Use of claim 6, wherein the calcineurin inhibitoris a compound of formula


12. Use of claim 6, wherein the calcineurin inhibitor is a compound offormula III wherein R is ethyl.
 13. Use of claim 6, wherein thecorticoid is selected from the group consisting of hydrocortisone,betamethasone and dexamethasone.
 14. Use of claim 6, wherein thecalcineurin inhibitor, the corticoid, or both, are in the form of asalt.
 15. A pharmaceutical composition comprising, besidepharmaceutically acceptable excipient, in combination a compound offormula I_(p) and hydrocortisone.
 16. A pharmaceutical compositioncomprising, beside pharmaceutically acceptable excipient, in combinationa compound of formula I_(p) and betamethasone.
 17. A pharmaceuticalcomposition comprising, beside pharmaceutically acceptable excipient, incombination a compound of formula I_(p) and dexamethasone.